The tat trans-activators encoded by the known strains of primate immunodeficiency virus share a conserved, highly basic protein domain. Mutagenesis of this sequence in the tat gene of human immunodeficiency virus type 1 is shown here to reduce, but not eliminate, the trans-activation of human immunodeficiency virus type 1-specific gene expression. The degree of inhibition is shown to vary in a dose-dependent manner and is most marked at low levels of tat expression. Multiple mutations of the basic domain of tat were found to impair both the in vivo stability and the nuclear localization of the tat protein. It is proposed that this protein domain serves to efficiently target the tat gene product to its appropriate site or substrate within the nucleus of expressing cells.